Transmission electron microscopy of Plum pox virus, Prunus necrotic ringspot virus, Prune dwarf virus in plum (Prunus domestica, L.

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1 Volume 15(1), , 2011 JOURNAL of Horticulture, Forestry and Biotechnology Transmission electron microscopy of Plum pox virus, Prunus necrotic ringspot virus, Prune dwarf virus in plum (Prunus domestica, L.) Jakab-Ilyefalvi, Zs. 1, Pamfil D., 1 Craciun C. 2 1 University Of Agricultural Sciences And Veterinary Medicine, Department Of Biotechnology, Cluj- Napoca; 2 Babes-Bolyai University, Department Of Biology, Cluj-Napoca *Corresponding author. zsolt.jakab@yahoo.com Abstract Transmission electron microscopy (TEM) was used in order to detect the presence of Plum pox virus, Prunus necrotic ringspot virus and Prune dwarf virus particles in plum ( Prunus domestica,l.) shoots and leaves. The presence of virus particles was confirmed by DAS-ELISA serological technique. Filamentous particles approximately 750 nm long were found in ultrathin sections of leaves, shoots in plum morphological analysis of the viral particles confirmed the presence of the studied virus particles. Spheric-concentrical particles were found in Renclode Althan cultivars infected with Prunus necrotic ringspot virus. DAS-ELISA serological confirmed the presence of the virus and morphological analysis carried out with the help of TEM microscopy confirmed that the viral particles had the characteristics reviewed by other researchers. Also spheric-concentrical particles were found in leaves and shoots of Renclode Althan plum cultivar. The TEM aided morphological analysis and measurements together with DAS ELISA serological technique confirmed the presence of Prunus necrotic ringspot virus and Plum dwarf virus. No virus particles were found in ultrathin sections of growth tips in the analyzed plum cultivars of 0.2-0,4 mm diameter. Meristematic tissues does not contained the three viruses, confirming the fact that due to the high division rates at the apical dome, the multiplication of the virus is suppressed and do not reaches the central zone of the meristem (CZ). Embryonic cells of growth tips of plum up to 0,1 mm in diameter are PPV, PNRSV, PDV free in the analyzed blocks. Therefore it highly recommended that the meristem diameter for virus elimination should be 0,1-0,2 mm in order to achieve potential virus free plants. Key words transmission electron microscopy,virus,ppv,pnrsv,pdv,cultivar,meristem Plum pox virus (PPV) An accurate detection and morphological analysis of several viruses are very important in the in vitro elimination procedures by tissue culture, in the nuclear stock material production of stone fruit species and the green biotechnology system (Pamfil,D.,2008). Plum pox virus is a member of the Potyviridae family, genus Potyvirus. The virus was identificated in Bulgaria in 1917, the farmers observed considerable economical losses ( Atanasoff,1932) in plum orchards and the phenomena was called sharka desease. The virus generally infects the plants from the Prunus genus ( plum,apricot,chery), destroys the fruits, these being unsuitable for marketing, the sugar content, acids, flavors, antocians, vitamin C is decreased. The average weight at tolerant cultivars decreases with 0,2-3,7 % and with 21,8-34 % in the case of sensible cultivars ( Jordovic and Janda 1963 after Krizbay,L.2004). The massive fall of the fruits can achieve 28,6-37,4% in case of apricot and in plum even %, the exterior and interior qualitys being strongly influenced by the virus titre. The viral genome is a positive(+) oriented monocatene RNA helix having a dimension of 10 kb with a single segment and at the extremity is found a poly A tail. The main protein product of the plum pox virus has a dimension of 355 kda and can be scindated with 3 proteases and as result there are 9 fully functional protein products. The serological proprieties of the virus are due to the capsidal protein (CP), there are numerous viral strains ( PPV-D,PPV-M,PPV-Elam, PPV Rec etc.). The viral diagram of the virus is R/1:3,0-3,5/5-6:E/E:S/* Thermal inactivation point (T=10 min.) is C, longevity in cellular sap is 1-50 days, final dilution Viral simptoms on the leaves are mosaic concentric zones, decoloration of the leaves, and the 120

2 deformation of fruits. In order to eliminate this infectious virus there is necessary to regenerate new virus free plants starting from meristem cells taken from the terminal apexes of young plants using propper culture media, plant hormones, acclimation technique (Jakab,Zs. et al.,2008,). Prunus necrotic ringspot virus ( PNRSV) The prunus necrotic ringspot virus ( PNRSV) represents the most important virus after the plum pox virus, due to the negative effect in production.for the first time it was identificated and characterized in apricot and plum trees. Subsequently was demostrated also at sweet cherry and sour cherry. High economical losses are observed in cherry infected commercial plantations and nurseries reducing the yield and the graft compatibility.the viral symptoms in the deseased plants are called ringspot diseases due to the concentrical lesions produced by the virus ( dereglation of the metabolism of the leaves in certain infected zones) ( Nemeth,M,1986). The shape of the virions are isometric, approximately 23 nm in diameter. The content of RNA is 16 %, the molar percent of the nucleotides is G-27% ; A-25%;C- 21%;U-27%) (Nemeth,Maria,1986). The virus is moderately imogenic ( Nemeth, M., 1986). The Prunus necrotic ringspot virus (PNRSV) belongs to the Bromoviridae family, Ilar virus group, and the viral genome is organized in three RNA segments. RNA 3 codes two polypeptides which respond for the migration of the virus ( ORF3) and the viral capsid ( ORF 3b). The protein responsible for vector transmission is named as putative movement protein. The viral capsid has de kda molecular weight, which can vary according to viral strains. Prune dwarf virus The prune dwarf virus (PDV) was described for the first time in USA under the name plum dwarfing virus. Together with prunus necrotic ringspot virus ( PNRSV) causes several problems to orchard farmers and nursery specialists. Cultivars infected with this virus can register a yield loss of % (Kegler,1977, after Nemeth, M., 1986 ). The PDV virus can infect together with PNRSV the young plants from the nursery or the tree in commercial plantations, mixed infections are evident and has been diagnosed by DAS ELISA and RT PCR molecular methods. The prune dwarf virus causes special problems in nursery where the success at grafting percent is decreased due to methabolical deregulation which influences directly the graft union. The negative effect is materialized as following: the infected individuals remain dwarfed,with little and corrugated leaves. The prune dwarf virus (PDV) is from the Bromoviridae family from Ilar genus ( Ilarviruses). The virus particles are isodiametric, spheric, having a T=3 icosahedrical symmetry having a nm diameter.several researchears are confirming a diameter of nm. Physical proprieties of the virus was established by Fulton (1970a), longevity in crude plant sap diluted with phosphate tampon hours, thermal inactivation point between 45 ºC 54 ºC, final dilution Genomic structure of the virus is similar with the PNRSV virus. The viruses belonging to the Bromoviridae family are containing a genom composed by 3 major segments, positive oriented (+), monocistronic, monocatenar RNA. Segments RNA1 and RNA2 are coding the proteins which are involved in the viral replication. RNA 3 segment is bicistronic and codes two polypeptides ( Krizbay,L.,2004 ).The viral capsid is expressed from a subgenomic RNA4 zone, collinear with RNA3 3 ` segment. Materials and Methods Plant material of meristematic origin was taken from terminal buds of Prunus domestica shoots. The shoots were taken from generally infected trees with PPV virus (Plum pox virus ),Prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), from the external part of the canopy. Apical meristems were cut out using an Olympus BX51 stereomicroscope and different sizes of inoculum were taken ( 0,4-0.7 mm). Crude plant sap was extracted by Homex (Bioreba) extraction equipment and standard Bioreba protocol for extraction from young shoots and leaves from the exterior and center part of the canopy. Transmission electron microscopy Fixing was done in a solution of 3 % glutaraldehyde, adjusted to ph 7.2 using 0.1 M phosphate buffer. The post-fixing procedure was performed using 1 % osmic acid. The post-fixed microsamples were dehydrated in acetone solutions and afterwards were embedded in Epon 812. The pre-final step was the sectioning procedure effectuated with the UC 6 Leica ultramicrotome and contrasted with uranyl acetate and lead citrate. The examination was carried out in a JEOL- JEM 1010, TEM electronmicroscope. 121

3 Fig. 1 Ultramicrotome Leica EM UC6 DAS ELISA serological technique The principle of the method is that from the antigenantibody-conjugate sandwich the enzyme part hydrolyzes with the substratum and in this way the color of these is changed into yellow. This color reaction can be detected through a photometer and the intensity of the color is directly proportional with the viral concentration. The sample can be considered infected if the value of extinction exceeds twice the average of the healthy samples. In order to carry out the detection procedure, it was used the BIOREBA classic DAS ELISA protocol according to the manufacturers instructions. Sampling was effectuated by Homex ( Bioreba) extraction equipment and the optical density values were measured by a TECAN optical photometer. Fig. 2 JEOL-JEM 1010, TEM electronmicroscope Results and Discussions The study of the PPV virus at the transmission electron microscope The viral particles are filamentous having an average diameter of nm and a diameter of nm (Fig.1-4). The viral particles have a filamentous, flexuous exterior shape with a clear modal length, there are organized singular or in bunches or agregates in cases of high viral concentrations. We also analysed a large number of electronmicrographs and the results showed that in the central zone of the meristem ( CZ) there were never observable these particles just in the infected leaves and shoots due to the high division rate of the meristems. Fig. 1 Filimentous PPV virions at plum cultivar Iulia - decreased viral concentration Fig. 2 Filimentous PPV virions at plum cultivar Iulia - medium viral concentration 122

4 Fig. 3. Filimentous PPV virions at plum cultivar Jubileu 50 high viral concentration In the crude plant sap extracted from young shoots of PPV infected plants the virions have a longevity of 3-4 Fig. 4. Filimentous PPV virions,, high viral concentration, cultivar Geta days, particle morphology results show that the virions have a flexuous shape. b) c) d) e) Fig. 5 PPV particle-aggregate in high concentration The study of the PNRSV virus at the transmission electron microscope Fig.6 PNRSV virions, spherical protein envelope Fig.7 PNRSV virions, spherical protein envelope cultivar Jubileu 50 - sample1 cultivar Jubileu 50 - sample 2 123

5 The studies revealed an isodiametrical shape of the PNRSV virions, the viral particles having a diameter of nm, according to other researchers publication, typical for the Ilarvirus group, Bromoviridae. The study of the PDV virus at the transmission electron microscope Fig.8 PDV-ILAR virions, spherical protein envelope The effectuated transmission electronmicroscopy analysis confirms the isodiametrical structure of the PDV viral particles, having a higher diameter than the PNRSV particles (25-35 nm) typical of those from the ILAR virus group. a) b) c) Fig. 9 Meristematic cells at plum (Prunus domestica,l) cultivar Jubileu 50 Analysing the large amount of TEM electronmicrographs effectuated in the meristematic dome at the peripheral and central zone (CZ,PZ), virus particles were not observed in this region, due to the high division rates which is documented and observable in Fig.9. Conclusions Filamentous particles of PPV or their aggregates were found in the leaves and shoots at plum cultivars Jubileu 50, Ivan, Geta approximately 750 nm long and having a diameter of 15 nm. Isometrical and circular virus particles of PNRSV and PDV were found also in shoots and leaves of plum cultivars also, having a diameter of nm in infected plum cultivar Renclode Althan.There were effectuated a large number of electronmicroscopical analysis but in the embryonic zone where are taking place high division processes of the meristematic cells, there were not found PPV,PNRSV or PPV particles. Analysing a large number of electromicrographs obtained by transmission electronmicroscopy the results show that growth apexes of 0,1 mm can be used in order to initiate meristem cultures to obtain virus free plants. Also virus free plants can be obtained from meristematic cells of plants infected with the studied viruses using meristematic cell domain of 0,1-0,2 mm in stone fruits because the central zone (CZ) of the meristem dome does not contain virus particles. Acknowledgments This work has benefited from financial support through the 2010 POSDRU 89/1.5/S/52432 project, ORGANIZING THE NATIONAL INTEREST POSTDOCTORAL SCHOOL OF APPLIED BIOTECHNOLOGIES WITH ON ROMANIAN BIOECONOMY project co-financed by the European Social Fund through the Sectorial Operational Programe Human Resources Development References 1.Atanosoff,D., 1932,Plum pox, A new disease, Yearbook of University of Sofia, faculty of Agriculture,11:

6 2.Craciun, C., 2005, Citologia generala, Ed., Risoprint, p Craciun,C., 1984, Ultrastructal investigations of Nuclear formations in Carnation Cells from Cultured Tissues,Cytologia 49: Jakab Zs.,Pamfil,D.,Doina Clapa,Fira,Al,2008, In vitro regeneration and meristem culture of Prunus domestica cultivars, Bulletin UASMV Cluj-Napoca, Horticulture, Vol.65. (1) Jakab Zs.,Ultrastructural investigation concertning nuclear formations encounterd in meristematic tissues in Prunus domestica, Annals of Romanian Society of Cell Biology, Vol XIV(1)-1009.Pag Krizbay,L.,2004,Dezvoltarea avansata a metodelor si sistemelor de analiza a materialului saditor la speciile samburoase,teza de doctorat,universitatea Corvinus,Budapesta,Ungaria. 7.Nemeth,Maria,1986,Virus,mycoplasma and rickettsia diseases of fruit trees,p Pamfil, D. 2002, Biotehnologii horticole, Editura academic press, Cluj Napoca, ISBN Pamfil,D.,2008, Agriculture from green revolution to green biotechnology, Proceeding of the XVII Croation Symposium on Agriculture, February 18-21, Opatja, Croatia,